published_or_final_version / Physiology / Master / Master of Philosophy

<p>Homing endonucleases are rare-cutting enzymes that cleave DNA at a site near their own location, preferentially in alleles lacking the homing endonuclease gene (HEG). By cleaving HEG-less alleles the homing endonuclease can mediate the transfer of its own gene to the cleaved site via a process called homing, involving double strand break repair. Via homing, HEGs are efficiently transferred into new genomes when horizontal exchange of DNA occurs between organisms.</p><p>Group I introns are intervening sequences that can catalyse their own excision from the unprocessed transcript without the need of any proteins. They are widespread, occurring both in eukaryotes and prokaryotes and in their viruses. Many group I introns encode a HEG within them that confers mobility also to the intron and mediates the combined transfer of the intron/HEG to intronless alleles via homing.</p><p>Bacteriophage T4 contains three such group I introns and at least 12 freestanding HEGs in its genome. The majority of phages besides T4 do not contain any introns, and freestanding HEGs are also scarcely represented among other phages.</p><p>In the first paper we looked into why group I introns are so rare in phages related to T4 in spite of the fact that they can spread between phages via homing. We have identified the first phage besides T4 that contains all three T-even introns and also shown that homing of at least one of the introns has occurred recently between some of the phages in Nature. We also show that intron homing can be highly efficient between related phages if two phages infect the same bacterium but that there also exists counteracting mechanisms that can restrict the spread of introns between phages. </p><p>In the second paper we have looked at how the presence of introns can affect gene expression in the phage. We find that the efficiency of splicing can be affected by variation of translation of the upstream exon for all three introns in T4. Furthermore, we find that splicing is also compromised upon infection of stationary-phase bacteria. This is the first time that the efficiency of self-splicing of group I introns has been coupled to environmental conditions and the potential effect of this on phage viability is discussed.</p><p>In the third paper we have characterised two novel freestanding homing endonucleases that in some T-even-like phages replace two of the putative HEGs in T4. We also present a new theory on why it is a selective advantage for freestanding, phage homing endonucleases to cleave both HEG-containing and HEG-less genomes.</p>

In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.

The homodimeric Ocr protein of bacteriophage T7 is a molecular mimic of a bent double-stranded DNA molecule ~24 bp in length. As such, Ocr is a highly effective competitive inhibitor of the bacterial Type I restriction modification (R/M) system. Thus, Ocr facilitates phage infection of the bacterial cell to proceed unhindered by the action of the R/M defense system. The main aim of this work was to understand the basis of the DNA mimicry displayed by Ocr. The surface of the protein is replete with acidic residues, most or all of which mimic the phosphate backbone of DNA. Aspartate and glutamate residues on the surface of Ocr were either mutated or chemically modified in order to investigate their contribution to the tight binding between Ocr and the EcoKI Type I R/M enzyme. Single or double mutations of Ocr had no discernable effect on binding to EcoKI or its methyltransferase component (M.EcoKI). Chemical modification was then used to specifically modify the carboxyl moieties of Ocr, thereby neutralizing the negative charges on the protein surface. Ocr samples modified to varying degrees were analysed to establish the extent of derivatisation prior to extensive biophysical characterisation to assess the impact of these changes in terms of binding to the EcoKI R/M system. The results of this analysis revealed that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by at least ~800-fold. In addition, based on the known 3-D structure of the protein, a set of multiple mutations were introduced into Ocr aimed at eliminating patches of negative charge from the protein surface. Specifically, between 5 and 17 acidic residues were targeted for mutation (Asp and Glu to Asn and Gln, respectively). Analysis of the in vivo activity of the mutant Ocr along with biophysical characterisation of the purified proteins was then performed. Results from these studies identified regions of the Ocr protein that were critical in forming a tight association with the EcoKI R/M system. Furthermore by comparing the relative contribution of different groups of acidic residues to the free energy of binding, the actual mechanism by which Ocr mimics the charge distribution of DNA has been delineated.

Résumé: De nos jours, les bactériophages (c.-à-d. des virus bactériens, ou phages) sont reconnus comme un des principaux facteurs qui influencent l’évolution et la biologie bactérienne. De plus, la nature dynamique des relations phage-hôte engendre des adaptations mutuelles au niveau des stratégies d’infection et de défense, phénomène communément appelé « course à l’armement ». Malgré une importance démontrée chez de nombreuses espèces bactériennes, l’étude du rôle des phages dans la biologie du pathogène Clostridium difficile est demeurée très limitée. Or, les infections à C. difficile sont considérées comme étant la principale cause des diarrhées associées à la prise d’antibiotiques. Alors, l’objectif de la présente étude avait pour but de mieux caractériser l’implication des phages dans la biologie de C. difficile. Des travaux préalables ont montré que la lysogénisation par le phage tempéré φCD38-2 pouvait mener à une augmentation de la production de toxines chez certaines souches de C. difficile suggérant une implication des phages dans la virulence bactérienne. En utilisant cette étude comme point de départ, nous avons évalué l’influence de la lysogénisation du phage φCD38-2 sur le transcriptome global d’une souche de C. difficile d’importance clinique. Ainsi, nous avons montré que la lysogénisation par le phage φCD38-2 a un impact significatif sur la transcription de 39 gènes bactériens dont près de la moitié encodent des protéines reliées au métabolisme des sucres, suggérant une implication du phage dans les processus métaboliques de l’hôte. Cependant, le gène présentant la plus grande altération transcriptionnelle encode une protéine de surface nommée CwpV. À partir de sa localisation sur la surface bactérienne, nous avons démontré que son expression a un effet protecteur sur les cellules face aux infections par les phages. Les expériences subséquentes ont permis de lier l’activité antiphage au domaine carboxy-terminale variable de la protéine. Étant donné que l’adsorption virale n’est pas affectée par la présence de CwpV, nous avons établi que le mode d’action du système consiste à bloquer l’injection d’ADN virale dans la cellule bactérienne. De plus, l’effet antiphage est plus prononcé envers les siphophages comparé aux myophages suggérant un mode d’action dépendant de la morphologie virale. Finalement, les expériences préliminaires suggèrent que les cellules qui expriment la CwpV ont un avantage sélectif par rapport aux cellules qui ne l’expriment pas dans un essai de co-culture soumise à une infection virale. / Abstract: Bacteriophages (or simply phages) are viruses that specifically infect and kill bacteria. They are omnipresent in every niche where bacteria thrive and as such are considered as the most abundant biological entities in the biosphere. Their massive impact on bacterial biology has incited scientific community to consider the phages as the major driving force in bacterial evolution. Nowadays, it is also assumed that phages act as the principal vectors for horizontal transfer of genetic information among prokaryotes. Moreover, highly dynamic nature of phage host relationships usually results in mutual adaptations that effectively stimulates acquisition of new offensive and defensive strategies. This phenomenon is generally described as the “phage-host arms race”. Despite their obvious importance, the contribution of phages to the biology of Clostridium difficile, the main cause of nosocomial infectious diarrhea, has not been extensively explored. Thus, the main objective of this study was to assess the overall impact of phages to C. difficile lifestyle. Our previous work has revealed the potential of a specific C. difficile phage termed φCD38-2 to stimulate the production of bacterial toxins. Based on those results, we have performed a global study of the impact of the φCD38-2 lysogeny on the bacterial transcriptome. Thus, we have found a total of 39 genes whose expression was altered during the lysogeny of φCD38- 2 with near half of them encoding proteins implicated in bacterial sugar metabolism. This suggests phage implication in the regulation of bacterial utilization of carbon sources. However, the largest transcriptional alteration has been observed for cwpV which encodes a phase-variable surface-anchored protein. Owing to its variable nature, we have hypothesized that CwpV might play a role in phage infection and indeed, we have shown that CwpV expression protects bacterial cells from phage infection. Moreover, variable C-terminal domain of CwpV was found to be essential for antiphage phenotype since its deletion restored bacterial susceptibility to infection. Additionally, CwpV did not significantly affect phage adsorption, but phage DNA replication was prevented suggesting that CwpV act as a superinfection exclusion system. Interestingly, the antiphage effect was more pronounced against phages from Siphoviridae family compared to phages from Myoviridae family suggesting that structural differences are important for the antiphage phenotype. Finally, our preliminary data suggest that CwpV expression confers selective advantage when mixed cocultures are challenged by phage infection.

Streptococcus Agalactiae est depuis les années 1990, responsable d'infections invasives émergentes chez l'adulte. Nous montrons queles souches responsables de ces infections appartiennent majoritairement aux sérotypes V et Ia et aux deux clones phylogénétiquement éloignés, CC1 et CC23. L'étude du contenu prophagique montre une lysogénie fréquente suggérant l'importance de la lysogénie dans la spécialisation de ces souches particulièrement aptes à infecter l'adulte. Dans un deuxième temps, nous avons isolé sept phages tempérés de souches associées à des infections cutanées et ostéo-articulaires. Ces phages appartiennent à la famille des SIPHOVIRIDAE. L’analyse par restriction enzymatique de l’ADN phagique et l’amplification par PCR de fragments d’ADN prophagique a montré la diversité de ces phages et leurdifférence des phages isolés de souches associées aux infections materno-foetales. Les phagesisolés de souches lysogènes de CC1 ont présenté un spectre lytique étendu aux souches de tous les clones intra-species. / Streptococcus agalactiae has emerged since 1990 in infections in nonpregnant adults, We showed that the strains isolated from adult infections were mainly of serotypes V and Ia., and mainly belonged to the two phylogenetically distant clones, CC1 and CC23. The prophagic content study showed a frequent lysogeny, suggesting a role of lysegeny in the specialization of these strains able to infect adult. Also, we isolated seven phages from strains associated with cutaneous and osteoarticular infections in adult. Ces phages classified among SIPHOVIRIDAE. Restriction analysis of phagic DNA and PCR for prophagic DNA showed genetiacally diverse phages, distinct from the phages isolated from strains responsible for materno-foetal infections. Phages isolated from lysogenic strains of CC1 had a wide lytic spectrum and were able to lyse strains belonging to all clones intra-species.

Background: Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. Results: Here we explore the role of recombination in both maintaining and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra-and inter-lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. Conclusions: These findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.

Acquisition of a superantigen pathogenicity island (SaPI) significantly increases virulence in Staphylococcus aureus. Horizontal transfer of SaPIs occurs at high frequency and depends upon a helper bacteriophage, either through direct infection or SOS-mediated induction of a lysogen. SaPIs hijack the packaging machinery of the helper phage, leading to the formation of SaPI-containing transducing particles that can introduce the pathogenicity island into neighboring SaPI-negative cells. All SaPIs contain a conserved core of genes, some of which are co-transcribed as an operon and encode functions involved in helper exploitation. The goal of this study was to more fully understand the intricate relationships between the SaPI elements and their helper bacteriophages, specifically any regulatory crosstalk that might occur between them. We demonstrated phage-host interactions in 80 and 80α, and SaPI1 and SaPIbov1-mediated crosstalk with helper phage 80α. The phage Sri protein was shown to be a bi-functional protein that both derepresses SaPI1 and interferes with host chromosome replication. Incoming SaPI1 experiments showed that SaPI1 modulates the levels of the N-terminal part of orf14 mRNA. Induction experiments using the 80α ΔrinA phage as a genetic tool, reveal several new phage genes that SaPI1 targets for expression modulation. Finally, a novel SaPI1 interference mechanism was identified. In an 80α ΔrinA mutant, which cannot activate its late operon, SaPI1 can directly turn on expression of the packaging and structural genes in a noncanonical manner, initiating from the 2nd gene in the operon, the large terminase subunit.

The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development.

The emergence of genomics as a discrete field of biology has changed humanity’s understanding of our relationship with bacteria. Sequencing the genome of each newly-discovered bacterial species can reveal novel gene sequences, though the genome may contain genes coding for hundreds or thousands of proteins of unknown function (PUFs). In some cases, these coding sequences appear to be conserved across nearly all bacteria. Exploring the functional roles of these cases ideally requires an integrative, cross-species approach involving not only gene sequences but knowledge of interactions among their products. Protein interactions, studied at genome scale, extend genomics into the field of interactomics. I have employed novel computational methods to provide context for bacterial PUFs and to leverage the rich genomic, proteomic, and interactomic data available for hundreds of bacterial species. The methods employed in this study began with sets of protein complexes. I initially hypothesized that, if protein interactions reveal protein functions and interactions are frequently conserved through protein complexes, then conserved protein functions should be revealed through the extent of conservation of protein complexes and their components. The subsequent analyses revealed how partial protein complex conservation may, unexpectedly, be the rule rather than the exception. Next, I expanded the analysis by combining sets of thousands of experimental protein-protein interactions. Progressing beyond the scope of protein complexes into interactions across full proteomes revealed novel evolutionary consistencies across bacteria but also exposed deficiencies among interactomics-based approaches. I have concluded this study with an expansion beyond bacterial protein interactions and into those involving bacteriophage-encoded proteins. This work concerns emergent evolutionary properties among bacterial proteins. It is primarily intended to serve as a resource for microbiologists but is relevant to any research into evolutionary biology. As microbiomes and their occupants become increasingly critical to human health, similar approaches may become increasingly necessary.